Alloxan compounds as antibacterial and antifungal agents

ABSTRACT

Alloxan, alloxan monohydrate, and alloxan tetrahydrate can be used to inhibit and/or prevent the growth of undesirable herbs, bacteria, fungi, yeast, and other microorganisms. This invention is particularly concerned with the bacteriostatic and bactericidal properties of alloxan compounds against Staphylococcus aureus and Escherichia coli.

United States Patent 91 Apr. 17, 1973 Douros, Jr. et al.

ALLOXAN COMPOUNDS AS ANTIBACTERIAL AND ANTIFUNGAL AGENTS Inventors: John D. Douros, Jr.; A] Fred Kerst,

both of Littleton, Colo.

The Gates Rubber Denver, Colo.

Filed: Aug. 14, 1970 Appl. No.: 75,311

Assignee: Company,

U.S. Cl. ..424/251, 71/74, 71/92,

260/45.75 Int. Cl. ..A0ln 9/00, AOln 9/22 Field of Search ..424/251 References Cited OTHER PUBLICATIONS Bruckmann et al., J. Biol. Chem. 168,241 (1947).

Jacobs, Hypoglycemic Action of Alloxan, Proc. Soc. Exter. Cio. Med. 37; 407-409 (1937).

Primary ExaminerAlbert T. Meyers Assistant ExaminerAllen J. Robinson Attorney-Raymond Fink, H. W. Oberg, Jr. and Curtis H. Castleman, Jr.

[57] ABSTRACT 24 Claims, N0 Drawings BACKGROUND OF THE INVENTION The present invention relates to the use of alloxan compounds, i.e., compounds of the ureide of mesooxalic acid (alloxan) having the formula:

for killing or inhibiting the growth of such microorgan isms as bacteria, yeast, and fungi. The chemical literature reveals few subjects, even within the rapidly expanding discipline of biochemistry, which have generated so many patent applications as the areas involving the biochemical uses of compounds which contain the barbituric acid nucleus,

where R represents various substituents. Representative biochemical uses of such compounds are found inv US. Pat. Nos. 2,561,688; 2,605,209; 2,725,380; 2,820,035; 2,887,487; and 3,102,072.

One reason for the tremendous range of applicationv metabolism, and potential toxic hazards of such microorganism inhibiting compounds along with their chemical structures and reactions. The alloxan, alloxan monohydrate, and alloxan tetrahydrate, of the present invention are no exception. References to some of the biological considerations associated with the use of alloxan compounds may be found in: Jacobs, I'I.R., Hypoglycemia Action of Alloxan, Proc. Soc. Exter. Cio. Med.,37: 407-409, (1937); Bailey, CC. and Bailey, O.T., Alloxan Diabetes with Diabetic Complications,Ne w Eng. J.Med., 230: 533-536,( 1944).

In addition to these biological applications, the patent literature, US. Pat. No. 3,105,075, also reveals that substituted alloxan compounds have been used to treat cellulosic textiles in order to make them resistant to creasing and wrinkling.

SUMMARY OF THE INVENTION According to the present invention, it has been found that the alloxan compounds:

' Alloxan, Alloxan Monohydrate, and Alloxan Tetrahydrate when present in effective quantities are very effective bactericidal, herbicidal, and fungicidal agents. References to various synthetic procedures for preparing alloxan are given by: Hartman, W. and Sheppard, 0., Org. Syn. Coll. Vol. III, 37 (1955); Sperand, J. and Daboirch, T., Org. Syn. Coll. Vol. III, 39 (1955); and Holmgren, A. and Wenner, W., Org. Syn. Coll. Vol. IV, 23 (1963).

The alloxan compounds of the present invention were prepared in the following manner: A 5-liter, 3- neck flask was fitted with an overhead stirrer, thermometer, and powder additional funnel. The reaction vessel was immersed in a cold water vessel. To the reaction vessel was added 1.2 liters of water, 1.2 kilograms of concentrated HCL, and 600 grams of uric acid. The mixture was rapidly stirred while grams of potassium chlorate were added over a l-hour period at a rate which kept the reaction mixture between 33 and 35 C. After the potassium chlorate was added, the reaction mixture became homogeneous. This solution was transferred to a 4-liter beaker and diluted to 3.6 liters with acetic acid. Upon cooling to 5 C. for several hours, an alloxan compound which was predominantly alloxan monohydrate precipitated. The alloxan monohydrate was characterized by its decomposition point (240 to 250C), infrared spectrum, and nitrogen analysis. The tetrahydrate was obtained by recrystallizing the monohydrate from'water. The anhydrous alloxan was prepared by subliming the alloxan monohydrate.

' The applicants have discovered that such alloxan compounds can be'used to inhibit the growth of a wide variety of bacteria and fungi. For example, alloxan compounds maybe employed:

AGAINST BACTERIA Bacteria Disease Produced Pseudomonas aeruginosa Plant infections are caused by various pseudomonads.

Erwim'a carotovam Various species of Erwinia attack commercial crops of carrots, tobacco, potatoes, squash, et cetera.

Various species of Xanthomonas cause a variety of diseases in plants such as sugar cane, rice, sugar beets, cotton, walnuts, wheat, rye, barley, beans, et cetera.

Xanthomonas phaseoli AGAINST FUNGI Fungi Altemaria Fusarium Aspergillus Rhizopus The applicants have also found that when the alloxan compounds of this invention are applied to plant life during growth,. harvest, and storage, microorganism growth is substantially arrested with very little damage to' the general environment since alloxan compounds exhibit a high degree of biodegradability.

By plant life, it is meant those organisms including living plants, tubers, seeds, bushes, vegetables, and trees, and the like. Particularly prone to rot caused by fungi are various crops of great commercial importance such as tomatoes, melons, corn, peanuts, cucumbers, bananas and the like. Such losses occur both during growth and after harvest while these crops are in storage and transit. It should be noted that the applicants alloxan compounds are particularly effective in reducingthe loss to tomato crops caused by Bacterial Leaf Spot and Late Blight of Tomatoes. The effectiveness of these alloxan compounds against these particular diseases. was established by the following test results:

Compound: Aqueous alloxan Aqueous solution alloxan solution Activity: Foliar fungicide i vs. Bacterial Leaf vs. Late Spot of Tomato Blight of I Tomatoes Inhibition at 500 ppm: 69% 48% Inhibition I at 1000 ppm: 100% 98% Inhibition I at 1500 ppm: 100% I% Control: Maneb Maneb Control Concentration: 100 ppm I00 ppm NOTE: The alloxan compounds were sprayed on 25 tomato plants previously inoculated with Late Blight of Tomatoes (Phyrophrhora infectans) and 25 tomato plants previously inoculated with Bacterial Leaf Spot of Tomatoes (Xanthomomis desicatoria). Additional tomato plants growing in the same area and similarly infected with blight and leaf spot diseases were left untreated as controls. Subsequent examination of the treated plants and untreated control tomato plants revealed the above indicated degrees of control in the treated plants while most of the untreated control plants developed severe symptoms of Late Blight of Tomatoes and Bacterial Leaf Spot of Tomato.

.The effectiveness of the alloxan compounds as a herbicide has also been established. 'Test results show:

% Inhibition at Those skilled in the art will recognize that the scope of the instant invention should not be limited by the above examples. For instance, it'should be recognized that protectant, systemic, and eradicant procedures may provide detection of other fungicidal actions. Pathogens representative of Phycomycetes, Ascomycetes, Basidiomycetes and the Fungi Imperfecti may.

provide indices of other fungicidal activity. Further more, other appropriate host plants may establish other.

plant tolerance data. Additional pathogens and appropriate host plants may well afford additional oppor-" tunities to further define the degree and spectrum of the activity disclosed in this invention. Since no firm and then progressively judged in subsequent studies. A wide range of pathogens, representative of economically important diseases, can be used to help define the spectra of activity and to assurehigh degrees'of success under field conditions. The following disease organisms, crops, and reference standards may also be used in such evaluations:

Disease Disease Organism Reference Compound Powdery Mildew of Erysiphe Maneb, Cucumbers cichoracearum Karathane Leaf Rust of Wheat Puccinia rubigo-vera Maneb, Karathane Leaf Rust of Wheat Puccima rubigo-vera Plantvax Bacterial Leaf Spot Xanthomonas vesicatoria Streptomycin of Tomatoes Rice Blast Disease Pin'culana aryzae Blasticidin Downy mildew of Sugar Peronospora e schacrii Karathane Beet- 1 Downy mildew of Lima Phythphrhora phaseali Karathane B Bean Rust 7 Uromyces phareoli var. I

Typica Karathane Powdery mildew of Erysiphe graminis Karathane Wheat g Powdery mildew of Podosphaera Ieucotricha Karathane Apple Powdery mildew of Sphaerolheca pannosa,

, var. rosac Karathane Roses Powdery mildew of Erysiphe I cicharacearum Karathane Cantalope Leaf Spot of Wheat Helminlhosparium sarivum j Maneb Early Blight of Altem'an'a .rolam' Maneb Tomato Rice Foot Rot Gibberella fugikuroi Blasticidin Cercospora Leaf Spot Cercospora bericola Maneb of Sugar Beets Septoria Leaf Spot Seploria apiigraveolentis Maneb of Celery Apple Scab Vemuria inaequalis Cyprex Common Bacterial Xanrhomonas phaseoli Streptomycin, Blight of Bean I Sulfate The applicants recommend the use of in vivo procedures whenever possible to test alloxan compositions of this invention to demonstrate their efficacy under more realistic conditions. However, not all pathogens lend themselves to such techniques. In order to provide additional spectrum definitions, the followmg fruit-rotting, storage decay, and bacterial pathogensmay be tested by "in vitro" methods:

Brown rot of Stone Sclerotlnia fructicola Captan Fruits v t Grey mold of Fruit Botrytis cinerea Maneb and Vegetables Rhizopus Fruit and Rhizopu: nigriean: Maneb Vegetable Rot v Citrus Blue Mold Penicilliumitalicum Karsthane Citrus Green Mold Penicillium digimrum Maneb Blue Mold of Apple Penicilliuml'lxpamum Maneb Bacterial Disease on Puudomona: syringe: Captan many fruit crofis ct Erwim'a camrovam Captan rules of procedure'can be laid down for the sequence of such evaluations'orfor the choice of pathogens, each compound must be considered on the basis of its demonstrated performance in such primary evaluations Bacterial Soft Another important advantage of alloxan fungicides, particularly in their agricultural applications, is that they can be made up in solid or liquid formulations. Examples of solid formulations are dust, wetable powders, granules, and pellets. As a dust, alloxan compounds may be dispersed in powdered solid carriers such as talc, soaps, soapstone, attapulgus clay, as well as other finely divided solids known to the dusting art. When formulated as wetable powders, the active component may be employed in conjunction with interfillers which may be of the clay type carrier or non-clay type, in conjunction with various combinations of wetting agents and emulsifiers which permit the adaptation of the concentration as a free flowing powder.

Each of these carriers may contain one or more of the specified alloxans or unresolved mixtures of the alloxan compounds of this invention with other carriers or extenders which are ordinarily non-reacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates, and the like, as.

well as diatomacious earth, micas, or other suitable materials.

When liquid formulations are desirable, liquid extenders, dilutants or carriers of a non-reactive nature may be utilized. Examples of such materials are alcohol, ketones, glycols, aromatic hydrocarbons, petroleum fractions such as octane, and various other distillates.

Where it is desired to use the aforementioned wetable powders or liquid formulations, either emulsified, dispersed, or suspended in water or other fluids, one or more of the class of materials herein referred to as adjuvants can also be incorporated into the powder, dust, or liquid formulation. These adjuvants comprise surface active agents, detergents, wetable agents, stabilizer's, dispersing agents, suspending agents, emulsifying agents, spreaders, stickers, and conditioning agents generally. To their modifying characteristics these adjuvants may facilitate handling and application and infrequently enhance or potentiate the alloxan compositions of this invention in their inhibitory activity by mechanisms which are frequently not well understood. A satisfactory but not exhaustive list of these adjuvants appears in Soap Chemical Specialities, Volume 31No. 7, Page 61; No. 8, Pages 48 61; No. 9, Pages 52 67; and No. 10, Pages 38 67 (1955). See also, Bulletin No. 607 of the Bureau of Entomology and Plant Quarantine of the United States Department of Agriculture.

An additional advantage of such alloxan compositions is their compatability with a variety of other biocidal and fungicidal materials. For example, it may be convenient to combine one or more alloxan compositions with one or more adjuvants and carriers with other pesticides, biocides, and fungicides of various structures. For example, the alloxan fungicidal inhibitors may be combined with insecticidal materials such as chlordane, benzene hexachlorides, dichlorodiphenyl trichloroethane (DDT), the insecticidal carbamates, polychlorinated terpenes, parathions, methoxychlor, insecticidal phosphates, phosphorothioates, phosphorodithioates and with fungicides such as sulphur, quinones, dodecylgaunidine, and metal dimethyldithiocarbamates.

There are, however, other considerations, such as compound concentrations and methods of application, which may make some methods of application more favored than others. These considerations may include the type of organisms on which the compound is to be administered, the degree of activity, the degree of inhibition toward the organism, and side effects. Also to 1 be considered, is the cost of production and the characteristic solubility of the compounds into which the alloxan is to be introduced.

In their antibacterial aspects, the applicants have found the alloxan compounds of this invention, when present in effective quantities, act as bacteriocides at relatively low concentrations. For example, it has been discovered that alloxan compounds are especially efficacious against Staphylococcus aureus and Escherichia coli. The in vitro effectiveness of these alloxan compounds against these bacteria was established by the following test:

One loopful of each of the Staphylococcus aureus and Escherichia coli was transferred from agar slants to 10 ml. of trypticase soybroth and incubated at 37 for 18 hours. At the end of this time, the bacteria were seeded into the same medium (+2 percent agar) 19 which the original inoculum was prepared. The bacteria were seeded at 1 ml. of inoculum per 250 ml. of medium which were equivalent to at least 1 X 10 cells/ml. The resulting mixture was poured into a heat resistant petri dish at a temperature of 45 C Analytical filter paper discs of r; inch diameter were used for the agar diffusion technique. Each disc is saturated with 0.08 ml. of the solubilized test compounds at mg./disc and placed on the surface of the hardened agar. The plates are incubated at 37 C. for 18 hours. The activity of the alloxan compounds was established byv measuring the zone of inhibition in centimeters. The larger the zone of inhibition, the more potent the anti-bacterial activity of the alloxan compound. Untreated control plates were used as a basis for comparison and these exhibited a diffused, profuse growth of bacteria.

The test results using alloxan, alloxan monohydrate, and alloxan tetrahydrate against these bacteria and against other bacteria under similar experimental conditions produced the following results:

Microorganism Zone of Zone of Zone of inhibition ln Centimeters for Alloxan Tetrahydrate (GRAM positive inhibition inhibition and GRAM negative In Centiln Centibacteria) meters for meters for Alloxan Alloxan Monohy drate Staphylococcus aureus 1.8 1.6 1.3 ATCC No. 6538 Escherichia coli [.6 1.4 1.2 ATCC No. 9637 Shigella dysenteriae 0.6 0.5 0.4 ATCC No. 9212 Shigella .ronnei 0.6 0.5 0.4 ATCC No. 9290 Salmonella panama L0 0.9 0.7 ATCC No. 7378 Salmonella paralyphi L0 0.9 0.7 ATCC No. 928! Salmonella enteritis L0 0.9 0.7 ATCC No. 13076 Salmonella pullorum L0 0.9 0.7 ATCC No. 10398 Salmonella derby 1.0 0.9 0.7 ATCC No. 6960 Salmonella gallinarium 1.0 0.9 0.7 ATCC No. 9184 Salmonella typhosa 1.0 0.9 0.7 ATCC No. 19430 Neisseria gonorrhoeae 3.3 3.0 2.5 ATCC No. l9424 Neisseria weichselbaumii 3.2 2.9 2.5 ATCC No. Neisseria meningilldi: 3.2 2.9 2.4

Although the precise mode of action whereby alloxan compounds inhibit the growth of such bacteria is not completely understood, it is believed that the alloxan compounds of this invention may serve as chemical antagonists; that is, as chemicals which compete with enzymes essential to the development of such bacteria. Since enzymes perform their catalytic function by virtue of their affinity for their natural substrate; any compound resembling a substrate in its chemically critical aspect may also have an affinity for the enzymes. If this affinity is great enough, the analog will displace the normal substrate from the enzyme and will prevent the growth reaction from taking place. It is believed that alloxan has a chemical affinity for an essential site on one enzyme necessary for bacterial growth and life.

,To further illustrate the workings of anti-bacterial and anti-fungal aspects of this invention, the following other examples are submitted:

EXAMPLE 1 Determining Anti-Fungal Activity of Alloxan Compounds Against Test Organisms.

Experimental The following viable test fungi are treated as described below:

ATCC No. 13077 Listeria monacytogenes 1.5 1.3 1.1 ATCC No. 15313 Vibrio fetus 2.0 1.8 1.5 ATCC No. 15296 Vibrio cholerne 2.0 1.8 1.5 ATCC No. 14035 Mycobacterium leprae 1.0 0.9 0.7 ATCC No. 4243 Mycobacrerium avium 1.0 0.9 0.7 ATCC No. 19421 Mycobacterium bovia 1.0 0.9 0.7 ATCC No. 19274 Mycobaclerium phlei 1.0 0.9 0.7 ATCC No. 11782 Micrococcus tetragena 1.0 0.9 0.7 ATCC No. 10875 Micrococcus melilensis 1.0 0.9 0.7 ATCC No. 19396 Micrococcus Iysodeikticus 1.0 0.9 0.7 ATCC No. 4698 Corynebacterium diphtheriae 2.2 2.0 1.6 ATCC No. 19409 Corynebacterium haemolyticum 2.2 2.0 1.6 ATCC No. 9345 Diplococcus intracellularir 2.2 2.0 1.6 ATCC No. Diplococcus pneumoniae 2.2 2.0 1.6 ATCC No. 6303 Haemophilus haemolyticus 2.2 2.0 1.6 ATCC No. 10014 Haemophilus influenzae 2.2 2.0 1.6 ATCC No. 19418 Haemophilus parainfluenzae 2.1 1.9 1.5 ATCC No. 7901 Haemophilus .ruis 2.1 1.9 1.5 AT CC No. 19417 Haemophilus vaginalis 2.2' 2.0 1.6 ATCC No. 14018 Bacleroides nigrescens 0.9 0.8 0.6 ATCC No. Bacteriodes pneumosinles 1.0 0.9 0.7 ATCC No. Bacteroides serpenr 1.0 0.9 0.7 ATCC No. Brucella abortus 1.0 0.9 0.7 ATCC No. 4315 Brucella melitensis 1.0 0.9 0.7 ATCC No. 19396 lirucella suis 1.0 0.9 0.7

ATCC No. 4312 F usarium oxysparum f usarium roseum Rhizopus nigricans Rhizopus stolonifer Aspergillus niger Alternaria solani One loopful of each of the above viable fungi cul- 'tures, spores, and mycelia is tranferred from an agar slant to an ml. portion of the nutrient broth composed of oatmeal agar, Czapeks, Sabouraud, and Deionized water to volume.

The 80 ml. portion of the fungi and broth is placed in a sterile shake flask (300 ml.) and the flask is placed on a rotary shaker for 96 to 120 hours at room temperature. At the end of this incubation time period, 10 ml. of the liquid are homogenized and placed into another sterile shake flask (300 ml.) containing 80 ml. of the above nutrient broth and 60 p.p.m. of the inhibitor being evaluated. The flasks are placed on a rotary shaker operating at 240 rpm. at room temperature for 3 to 9 days. After this second incubation time, the flasks are taken off and examined for visible fungal growth and mycelial weights are determined. Untreated controls are used as the basis of comparison.

RESULTS The alloxan compounds of this invention imparted a substantial degree of inhibition of fungal growth at 60 p.p.m.

EXAMPLE ll Formulation of a Paint Having Anti-Mildew Properties.

The following ingredients are blended and ground together in the indicated proportions in a ball mill.

Volume adjusted to 1000gallons by the addition of naphtha.

EXAMPLE lll Anti-Mildew Paint Formulation.

The following ingredients are blended together in the indicated proportions in a ball mill.

Pounds per Ingredient 1000 Gallons Rosin 300 Coal tar 100 Talc 100 Pine oil 50 Alloxan 50 High flash naphtha and mineral spirits 200' Volume adjusted to 1000 gallons by the addition of high flash naphtha and mineral spirits.

EXAMPLE IV Preparation of a Vinyl Coating Resistant to Mildew Deterioration.

A vinyl coating is prepared using a commercially available preparation without a fungal growth inhibitor.

An identical vinyl coating is prepared except that 2 percent by weight of alloxan monohydrate is incorporated into the coating formulation.

Two sets of components such as asbestos tubing, silkwrapped transformers and rayon-wrapped solenoids are obtained. One set is sprayed with the vinyl coating containing inhibitor, the other with the identical coating without inhibitor.

EXAMPLE V Preparation of Plasticizers Resistant to Mildew.

A commercial thermoplastic monomer is divided into four portions which are treated as follows:

Portion 1: To this first portion is added 2 percent by weight of alloxan monohydrate and percent by weight of dimethylnaphthalate as plasticizer. The monomer is polymerized and and molded as above.

Portion 2: To this portion is added 2 percent by weight of alloxan monohydrate and 10 percent by weight of butyl isodecylphthalate as plasticizer. The monomer is polymerized and molded as above.

Portion 3: This portion is the untreated control of Portion 1 containing no fungal inhibitor but 10 percent by weight of dimethylphthalate as plasticizer. Again, the polymerization and molding are identical.

Portion 4: This portion is the untreated control of Portion 2 containing no fungal inhibitor but 10 percent by weight of butyl isodecylphthalate as plasticizer. The polymerization and molding are as described above.

The two plasticizers are chosen on the basis of their known susceptibility to Fusarium attack under high humidity and temperature conditions.

EXAMPLE VI Evaluation of the Paint Formulations,

Vinyl Coatings, and Plasticizers for Mildew and Fungal Resistance.

A. The paint formulations from Examples II and ii] are painted on steel test panels which are allowed to dry and then placed in an air-tight high temperature and high humidity chamber maintained at 80 F. and 9 5 percent humidity to stimulate tropical temperature and humidity conditions. At the same time, steel test panels painted with the untreated control formulations are placed in the same chamber.

B. The vinyl coated articles of Example IV are placed in an identical heat and humidity chamber with control articles.

C. The four discs of Example V made as previously described are placed in a third high temperature and humidity chamber similar to the two described above.

RESULTS A. After a one-month test period, the control paint panels are found to be coated with various fungi including Fusarium, Penicillium, and Aspergillus species and are discolored. The treated painted panels are unaffected.

B. After a months exposure the vinyl coated articles treated with inhibitor are only slightly attacked by rot while the articles coated with vinyl without inhibitor are rotted through.

C. After a month of testing, the two untreated control discs are examined and are found to be blackened and rotted. Isolates of Aspergillus and F usarium of unknown species are prepared from the deteriorated discs. The two discs containing fungal inhibitors are not adversely affected.

EXAMPLE VII Evaluation of Alloxan Compositions as Mildew Retardant in Cotton.

Tests similar to those described in detail in Method 5762, Mildew Resistance of Cloth; Soil Burial Method, of Federal Specification CCC-T-l9l, Textile Test Methods are used; that is, standard blueline cotton duck fabric (3" X 3") is impregnated with 2 percent solutions of alloxan composition calculated on the dry weight of the fabric. The fabric is cut in three 1" X 3 strips which are raveled and buried vertically. At the same time, an identical piece of 3" X 3" cotton duck fabric untreated for control purposes is buried using the same techniques and location. At the end of 5 days burial, the two groups of buried cotton are removed and tested for loss of breaking strength as compared to unburied controls. Cloth treated with alloxan compounds requires substantially more force to break than the untreated control cloth.

Having thus disclosed our invention, we claim:

1. A method of killing or inhibiting the growth of microorganisms selected from the groupconsisting of fungi, Gram positive bacteria, and Gram negative bacteria which comprises applying to said microorganisms an effective fungicidal, bactericidal or bacteriostatic amount of an alloxan compound wherein said alloxan compound is selected from the group consisting of alloxan, alloxan monohydrate, alloxan tetrahydrate and mixtures thereof. 7

2. The method of claim 1 wherein the fungicidal, bactericidal or bacteriostatic compound is alloxan.

3. The method of claim 1 wherein the fungicidal, bactericidal or bacteriostatic compound is alloxan monohydrate.

4. The method of claim 1 wherein the fungicidal, bactericidal or bacteriostatic compound is alloxan tetrahydrate.

5. The method according to claim 1 wherein the Gram positive bacteria are selected from the group consisting of Staphylococci, Corynebacter, Listeria, Micrococci, Mycobacterium, and Diplococci.

6. The method according to claim 5 wherein the Staphylococci is Staphylococcus aureus.

7. The method according to claim 5 wherein the Corynebacter are selected from the group consisting of Corynebacterium diphtheriae and Corynebacterium haemolyticum.

8. The method according to claim 5 wherein the Listeria is Listeria monocytogenes.

9. The method according to claim 5 wherein the Micrococci are selected from the group consisting of Micrococcus tetragena, Micrococcus melitensis, and Microcaccus lysodeikticus.

10. The method according to claim 5 wherein the Mycobacterium are selected from thegroup consisting of Mycobacterium avium, Mycobacterium bovis, Mycobacterium phlei, and M ycobacterium leprae.

11. The method according to claim wherein the Diplococci are selected from the group consisting of Diplococcus .intracellularis and Diplococcus pneumoniae. v I

12. The method according to claim 1 wherein the gram negative bacteria are selected from the group consisting of Escherichia, Shigella, Salmonella, Vibrio, Neisseria, Haemophilus, Bacteroides, and Brucella.

13. The method according to claim 12 wherein the Escherichia is Escherichia coli.

14. The method according to claim 12 wherein the Shigella are selected from the group consisting of Shigella dysenteriae, and Shigella sonnei.

15. The method according to claim 12 wherein the Salmonella are selected from the group consisting of Salmonella derby, Salmonella enteritis, Salmonella gallinarium, Salmonella panama, Salmonella paratyphi, Salmonella pullorum, and Salmonella iyphosa.

16. The method according to claim 12 wherein the Vibrio are selected from the group consisting of Vibrio cholerae and Vibrio fetus.

17. The method according to claim 12 wherein the Neisseria are selected from the group consisting of Neisseria gonorrhoeae, Neisseria weichselbaumii, and Neisseria meningitides.

18. The method according to claim 12 wherein the Bacteroides nigrescens, Ba cteroides .pneumosintes, andv Bacteroides serpens.

20. The method according to claim 12 wherein the Brucella are selected from the group consisting of Brucella abortus, Brucella melitensis, and Brucella suis.

' 21. The method of claim 1 wherein the fungi is selected from the group consisting of Fusarium, Penicillium, and Aspergillus.

22. The method according to claim 1 wherein the bacteria is X anthomonas desicaioria.

23. The method according to claim 1 wherein the bacteria is Phytophthora infectans,

24. A method of killing or inhibiting the growth of microorganisms selected from the group consisting of fungi, Gram positive bacteria and Gram negative bacteria on plants which comprises applying to said plants an effective bactericidal, bacteriostatic or fungicidal amount of a compound selected from the group consisting of alloxan, alloxan monohydrate, alloxan tetrahydrate and mixtures thereof.

* l IF 

2. The method of claim 1 wherein the fungicidal, bactericidal or bacteriostatic compound is alloxan.
 3. The method of claim 1 wherein the fungicidal, bactericidal or bacteriostatic compound is alloxan monohydrate.
 4. The method of claim 1 wherein the fungicidal, bactericidal or bacteriostatic compound is alloxan tetrahydrate.
 5. The method according to claim 1 wherein the Gram positive bacteria are selected from the group consisting of Staphylococci, Corynebacter, Listeria, Micrococci, Mycobacterium, and Diplococci.
 6. The method according to claim 5 wherein the Staphylococci is Staphylococcus aureus.
 7. The method according to claim 5 wherein the Corynebacter are selected from the group consisting of Corynebacterium diphtheriae and Corynebacterium haemolyticum.
 8. The method according to claim 5 wherein the Listeria is Listeria monocytogenes.
 9. The method according to claim 5 wherein the Micrococci are selected from the group consisting of Micrococcus tetragena, Micrococcus melitensis, and Micrococcus lysodeikticus.
 10. The method according to claim 5 wherein the Mycobacterium are selected from the group consisting of Mycobacterium avium, Mycobacterium bovis, Mycobacterium phlei, and Mycobacterium leprae.
 11. The method according to claim 5 wherein the Diplococci are selected from the group consisting of Diplococcus intracellularis and Diplococcus pneumoniae.
 12. The method according to claim 1 wherein the gram negative bacteria are selected from the group consisting of Escherichia, Shigella, Salmonella, Vibrio, Neisseria, Haemophilus, Bacteroides, and Brucella.
 13. The method according to claim 12 wherein the Escherichia is Escherichia coli.
 14. The method according to claim 12 wherein the Shigella are selected from the group consisting of Shigella dysenteriae, and Shigella sonnei.
 15. The method according to claim 12 wherein the Salmonella are selected from the group consisting of Salmonella derby, Salmonella enteritis, Salmonella gallinarium, Salmonella panama, Salmonella paratyphi, Salmonella pullorum, and Salmonella typhosa.
 16. The method according to claim 12 wherein the Vibrio are selected from the group consisting of Vibrio cholerae and Vibrio fetus.
 17. The method according to claim 12 wherein the Neisseria are selected from the group consisting of Neisseria gonorrhoeae, Neisseria weichselbaumii, and Neisseria meningitides.
 18. The method according to claim 12 wherein the Haemophilus are selected from the group consisting of Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus suis, and Haemophilus vaginalis.
 19. The method according to claim 12 wherein the Bacteroides are selected from the group consisting of Bacteroides nigrescens, Bacteroides pneumosintes, and Bacteroides serpens.
 20. The method according to claim 12 wherein the Brucella are selected from the group consisting of Brucella abortus, Brucella melitensis, and Brucella suis.
 21. The method of claim 1 wherein the fungi is selected from the group consisting of Fusarium, Penicillium, and Aspergillus.
 22. The method according to claim 1 wherein the bacteria is Xanthomonas desicatoria.
 23. The method according to claim 1 wherein the bacteria is Phytophthora infectans.
 24. A method of killing or inhibiting the growth of microorganisms selected from the group consisting of fungi, Gram positive bacteria and Gram negative bacteria on plants which comprises applying to said plants an effective bactericidal, bacteriostatic or fungicidal amount of a compound selected from the group consisting of alloxan, alloxan monohydrate, alloxan tetrahydrate and mixtures thereof. 